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Background and Purpose The present treatment for choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD) is not sufficient. Hence, we examined the therapeutic efficacy of reducing histamine H4 receptor expression on CNV in mice. Experimental Approach H4 receptor expression was examined in CNVs from patients with AMD. In mice, laser photocoagulation was performed in the retina to induce experimental CNV (laser CNV). Protein and mRNA expression levels were determined and CNV volume measured in wild-type and Hrh4-/- mice with laser CNV. The effects of JNJ7777120, an H4 receptor antagonist, administered intravitreously, on CNV volume and pathological vessel leakage were determined in mice with laser CNV and controls. Fundus imaging, retinal histology and electroretinography were performed on eyes injected with JNJ7777120 to evaluate retinal toxicity. Key Results Human H4 receptors were only confirmed in CNV samples from AMD patients and not in the other subretinal tissues. Mouse H4 receptors were expressed in retinal pigment epithelium only after inducing laser CNV in wild-type mice, and were co-localized with the macrophage marker F4/80. Laser CNV volume was reduced in Hrh4-/- mice compared with that in wild-type mice, and JNJ7777120 suppressed laser-induced CNV volume and pathological CNV leakage in wild-type mice. Also eyes injected with JNJ7777120 did not show retinal degeneration. Conclusions and Implications H4 receptors are expressed in macrophages that accumulate around CNVs. Suppressing H4 receptor expression prevented the pathological vessel leakage without showing retinal toxicity, indicating that the H4 receptor has potential as a novel therapeutic target in AMD.

We fabricated conductive Al-doped ZnO (AZO) films on glass substrates via a simple drop-coating process of alcoholic dispersion solutions of AZO nanoparticles less than 10 nm in size, which were prepared by hydrolysis reactions of Zn(NO3)2·6H2O and Al(NO3)3·9H2O with an excess amount of isopropylamine. After heating at 150 °C to completely remove the alcoholic solvents, a by-product that remained, isopropylammonium nitrate, was melted and functioned as a low-temperature medium for the AZO nanoparticles. Even in the low-temperature medium at 150 °C, the AZO nanoparticles could readily grow up to ~100 nm, based on Ostwald ripening as a plausible crystal growth mechanism. The medium was evaporated at 240 °C, and a highly transparent AZO film appeared on the glass with a transmittance of ~83% in the visible region. The electrical conductivity of the AZO films was improved by sintering at 450 °C and post-annealing at 450 °C in a stream of a mixed gas of N2 and H2. The resistivity of the AZO film reached 4.3 × 10−2 Ω cm in an Al/Zn molar ratio of 2.0%.

Purpose Diabetic macular edema (DME) is characterized by an accumulation of fluid in the macula due to diabetic retinopathy. Currently, anti-VEGF drugs are the standard treatment worldwide for DME. This study aimed to assess whether the existence of epiretinal membrane (ERM) affects anti-VEGF efficacy, due to reduced permeability of the antibody through the ERM. Methods We retrospectively examined clinical data of DME patients who underwent anti-VEGF treatment and evaluated whether clinical differences existed between DME eyes with ERM and those without ERM. We then created an in vitro ERM model using MIO-M1, ARPE-19, and NTI-4 cells on Transwell membranes and evaluated antibody permeability through this in vitro ERM model using fluorescently labeled antibodies. Results Central retinal thickness (CRT) change between before and 1 month after first anti-VEGF treatment, as well as final CRT and final visual acuity 12 months after first anti-VEGF treatment, significantly differed between DME eyes with ERM and those without ERM. The in vitro ERM model led to production of collagen I in a manner similar to that of human ERM specimens. Fluorescence intensity of the lower chamber of the in vitro ERM model was significantly reduced in a dose-dependent manner. Conclusions Clinical data analysis indicated that the existence of ERM in DME eyes lowered the efficacy of anti-VEGF treatment. Reduced antibody permeability through the in vitro ERM model suggested ERM presence was associated with resistance to anti-VEGF treatment in DME eyes with ERM.

Purpose The misuse of inauthentic cell lines is widely recognized as a major threat to the integrity of biomedical science. Whereas the majority of efforts to address this have focused on DNA profiling, we sought to anatomically, transcriptionally, and functionally authenticate the RF/6A chorioretinal cell line, which is widely used as an endothelial cell line to model retinal and choroidal angiogenesis. Methods Multiple vials of RF/6A cells obtained from different commercial distributors were studied to validate their genetic, transcriptomic, anatomic, and functional fidelity to bona fide endothelial cells. Results Transcriptomic profiles of RF/6A cells obtained either de novo or from a public data repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers were very low or undetectable in RF/6A compared to primary human endothelial cells. Importantly, RF/6A cells also did not display functional characteristics of endothelial cells such as uptake of acetylated LDL, expression of E-selectin in response to TNF-α exposure, alignment in the direction of shear stress, and AKT and ERK1/2 phosphorylation following VEGFA stimulation. Conclusions Multiple independent sources of RF/6A do not exhibit key endothelial cell phenotypes. Therefore, these cells appear unsuitable as surrogates for choroidal or retinal endothelial cells. Further, cell line authentication methods should extend beyond genomic profiling to include anatomic, transcriptional, and functional assessments.

Purpose A retro-odontoid pseudotumor that is not associated with rheumatoid arthritis or hemodialysis is clinically rare. The majority of surgeons select transoral resection as the surgical treatment, often followed by posterior fusion or posterior decompression and fusion. In contrast, some authors have reported success with simple decompression without posterior stabilization in cases where atlanto-axial instability (AAI) is either absent or minor. In this study, we have evaluated the clinical and radiographic outcomes of C1 laminectomy without fusion as the surgical treatment for patients with cervical myelopathy that is associated with a retro-odontoid pseudotumor. Methods A retrospective chart review was conducted on 10 patients who underwent C1 laminectomy without fusion for cervical myelopathy associated with a retro-odontoid pseudotumor. Results The average follow-up time was 29 months. All cases were graded as Ranawat grade 3a or 3b. After surgery, myelopathy improved in all of the patients. In 2 patients, the atlas-dens interval increased in the flexed position; however, this did not result in any clinical problems. The size of the retro-odontoid mass (measured on magnetic resonance images at least 12 mo after surgery) decreased in 4 of the 10 cases. Conclusions AAI progression and mass enlargement were our primary concerns for this surgical option; however, C1 laminectomy did not cause severe AAI progression, no patients showed serious mass enlargement, and all patients demonstrated neurological improvement. This surgical strategy is beneficial especially for elderly patients given the risks of other surgical options that use an anterior transoral approach or posterior fusion.

Objective To examine and expand the genetic spectrum of Waardenburg syndrome type 1 (WS1). Methods Clinical features related to Waardenburg syndrome (WS) were examined in a five-year old patient. Mutation analysis of genes related to WS was performed in the proband and her parents. Molecular modeling of EDNRB and the p.R319W mutant was conducted to predict the pathogenicity of the mutation. Results The proband showed sensorineural hearing loss, heterochromia iridis, and dystopia canthorum, fulfilling the clinical criteria of WS1. Genetic analyses revealed that the proband had no mutation in PAX3 which has been known as the cause of WS1, but had a homozygous missense mutation (p.R319W) in endothelin receptor type B ( EDNRB ) gene. The asymptomatic parents had the mutation in a heterozygote state. This mutation has been previously reported in a heterozygous state in a patient with Hirschsprung’s disease unaccompanied by WS, but the patient and her parents did not show any symptoms in gastrointestinal tract. Molecular modeling of EDNRB with the p.R319W mutation demonstrated reduction of the positively charged surface area in this region, which might reduce binding ability of EDNRB to G protein and lead to abnormal signal transduction underlying the WS phenotype. Conclusions Our findings suggested that autosomal recessive mutation in EDNRB may underlie a part of WS1 with the current diagnostic criteria, and supported that Hirschsprung’s disease is a multifactorial genetic disease which requires additional factors. Further molecular analysis is necessary to elucidate the gene interaction and to reappraise the current WS classification.

To compare optical coherence tomographic angiography (OCTA) and indocyanine green angiography (ICGA) images for detecting polypoidal lesions (PLs) and branching vascular networks (BVNs), and to measure the polypoidal areas (PAs) in patients with polypoidal choroidal vasculopathy (PCV). All patients underwent ICGA, optical coherence tomography (OCT), and OCTA. We compared the detection sensitivity for PL and BVN, as evaluated by the ICGA and OCTA images. Furthermore, PA measured by ICGA was divided into two groups: one in which the area could be measured by OCTA (ICGA+OCTA+) and the other in which the area could not be measured by OCTA (ICGA+OCTA−). Twenty-one consecutive eyes of 21 patients (mean age, 73.8±9.8 years) were included. ICGA detected PL in all eyes (100%), whereas OCTA detected PL in 16 eyes (75.2%); ICGA detected BVN in 15 eyes (71.4%), whereas OCTA detected BVN in 20 eyes (95.2%). The mean PA in ICGA+OCTA+ and ICGA+OCTA− was 0.24±0.04 and 0.14±0.01 mm2, respectively; a significant difference was observed between ICGA+OCTA+ PA and ICGA+OCTA− PA (P

To evaluate, using pupillography, the difference between eyes affected by age-related macular degeneration and their contralateral normal eyes with regard to the mean relative afferent pupillary defect (RAPD) score. Also, to ascertain any correlations between this difference in RAPD score and differences in visual acuity or age-related macular degeneration (AMD) dimensions. Measurements were made using the RAPDx pupillographer (Konan Medical, Nishinomiya, Japan), which analyzes pupil response to light stimulation. Both best corrected visual acuity (converted to logMAR) and greatest linear dimension (GLD; calculated on the basis of fluorescence angiography images) were measured. The correlations between RAPD difference and logMAR difference, and GLD difference were then analyzed. The study included 32 patients (18 men, 14 women; mean age = 74.8 ± 9.7 years) who had AMD in 1 eye and a normal fundus in the contralateral eye. Mean resting pupil diameter, mean latency onset of constriction, mean velocity of constriction, and recovery were not significantly different in AMD eyes compared with normal eyes. The mean amplitude of constriction was smaller (P = 0.028), and the mean latency of maximum constriction was shorter (P = 0.0013) in AMD eyes than in normal eyes. Regarding RAPD scores, there was a significant correlation between visual acuity difference and RAPD score differences of both amplitude (P

Purpose We report a newly developed device to fragment thrombi in retinal vein occlusion. Methods The new instrument consists of a 23-gauge (G) pipe and a 37-G needle with an internal wire. A total of 40 porcine eyes were used; 20 eyes for experiments in the branch retinal vein (BRV group) and 20 eyes for experiments in the central retinal vein (CRV group). We placed 25-G 3-port trocars, and core vitrectomy was performed. Another 23-G scleral incision was performed for insertion of the needle. The needle pierced the retinal vein at a distance of three- to four- or one-disc diameters from the optic disc (BRV or CRV group, respectively), and the internal wire was advanced toward the disc. The success rates of needle piercing and cannulation of the internal wire were recorded in each group. In the CRV group, the cannulation was deemed successful when the tip reached inside the optic disc. Real-time optical coherence tomography imaging also was performed using the Zeiss Rescan 700 device in porcine eyes. Histologic examination of the retinal vessel inserted with the internal wire was performed. Results The success rates of needle piercing into the BRV and CRV were 85% and 95%, respectively. The success rates of cannulation of the internal wire into the BRV and CRV were 85% and 0%, respectively. The process of cannulation was recorded successfully with the Rescan 700. Histologic examination showed no damages to the endothelial cell layer. Conclusions The needle and internal wire intended to be used for recanalization of BRV occlusion were successfully pierced and cannulated into the BRV. Translational relevance This newly developed device could become a treatment modality for retinal vein occlusion to fragment thrombi that present treatment methods cannot reach and remove directly.

Purpose: We compared 1-year outcomes of 1 + pro re nata (PRN) versus 3 + PRN of intravitreal aflibercept injection (IAI) for age-related macular degeneration (AMD). Methods: Forty-two eyes with naïve AMD received 3 + PRN IAI treatment and 47 eyes with naïve AMD received 1 + PRN IAI treatment. Visual acuity (VA), central retinal thickness (CRT), and central choroidal thickness (CCT) and number of administered IAIs during 12 months were compared. Results: VAs improved, and CRTs reduced significantly at any given month from baseline in both groups (p < 0.01, respectively). CCT reduced significantly at 3 months in the 3 + PRN group (p = 0.024) but not in the 1 + PRN group. The 1 + PRN group received fewer injections than the 3 + PRN group (p < 0.01). Conclusions: Aflibercept leads to equivalent VA and morphologic retinal improvement without administering 3 injections.

To compare short-term outcomes of intravitreal aflibercept injection (IAI) with or without initial photodynamic therapy (PDT) for polypoidal choroidal vasculopathy (PCV) using focal macular electroretinography (FMERG). Observation case series. Twelve patients (6 males, 6 females; 12 eyes) with naive PCV received 3 initial IAIs and a single session of PDT 3 days after the first IAI (combination group), and 13 patients (7 males, 6 females; 13 eyes) with naive PCV received 3 initial IAIs only (IAI group) were retrospectively observed. Changes in visual acuity, central retinal thickness (CRT), central choroidal thickness (CCT), and FMERG parameters (FMERGs) were compared. The combination group showed improved visual acuity after the second and third IAI (P = 0.040, 0.019, respectively); both groups showed reduced CRT after the first IAI (P

Purpose The purpose of this study was to determine microRNA expression in vitreous and subretinal fluid (SRF) samples from patients with retinal detachment (RD). The pathological importance of the identified microRNA transcript levels was analyzed in vitro. Methods Vitreous fluid was collected from 10 patients with macular hole (MH), vitreomacular traction syndrome (VMTS), or foveoschisis and from 11 patients with RD. Subretinal fluid was collected from 7 patients with RD. Of these, blood serum was collected in 4 patients. MicroRNA microarray profiling was performed to identify microRNA transcripts that were present in vitreous fluid, and more redundantly detected in SRF, of patients with RD, but not detected in control eyes. Western blotting and scratch assays were performed in ARPE-19 cells and primary human RPE cell lines transfected with microRNA to elucidate the effect of identified microRNA transcripts on epithelial-mesenchymal transition (EMT). Results MicroRNA microarray profiling revealed that hsa-miR-148a-3p was the most redundantly detected transcript in SRF and vitreous fluid from patients with RD, but not those with the other diseases. Expression levels of hsa-miR-148a-3p were higher in SRF samples than in blood serum samples in 3 out of 4 patients. Following hsa-miR-148a-3p mimic transfection, ARPE-19 and human RPE cells demonstrated increased expression of α-smooth muscle actin by Western blotting and increased migration ability during scratch assays. Conclusions The results of the present study indicate that hsa-miR-148a-3p was specifically detected in RD and promotes EMT in RPE.

Hypothesis Different missense mutations of the optic atrophy 1 gene (OPA1) identified in optic atrophy patients with auditory neuropathy spectrum disorder (ANSD) induce functional impairment through different molecular mechanisms. Background OPA1 is the gene responsible for autosomal dominant optic atrophy (ADOA), but some of its mutations are also associated with ANSD. OPA1 is a member of the GTPase family of proteins and plays a key role in the maintenance of mitochondrial activities that are dependent on dimer formation of the protein. There are many reports of OPA1 mutations, but the molecular mechanisms of their functional impairments are unclear. Methods The sequences of coding regions in OPA1 were analyzed from blood samples of ADOA patients with ANSD. Molecular modeling of the protein's ability to form dimers and its GTP-binding ability were conducted to study the effects of structural changes in OPA1 caused by two identified mutations and their resultant effects on protein function. Results Two heterozygous mutations, p.T414P (c.1240A>C) and p.T540P (c.1618A>C), located in the GTPase and middle domains of OPA1, respectively, were identified in two patients. Molecular modeling indicated decreased dimer formation caused by destabilization of the association structure of the p.T414P mutant, and decreased GTP-binding caused by destabilization of the binding site structure in the p.T540P mutant. Conclusion These two different conformational changes might result in decreased GTPase activities that trigger ADOA associated with ANSD, and are likely to be associated with mild clinical features. Molecular modeling would provide useful information in clinical practice.

Purpose. It is a matter of increasing concern that exposure to light-emitting diodes (LED), particularly blue light (BL), damages retinal cells. This study aimed to investigate the retinal pigment epithelium (RPE) damage caused by BL and to elucidate the role of nuclear factor (erythroid-derived)-related factor 2 (Nrf2) in the pathogenesis of BL-induced RPE damage.Methods. ARPE-19, a human RPE cell line, and mouse primary RPE cells from wild-type andNrf2knockout (Nrf2−/−) mice were cultured under blue LED exposure (intermediate wavelength, 450 nm). Cell death rate and reactive oxygen species (ROS) generation were measured. TUNEL staining was performed to detect apoptosis. Real-time polymerase chain reaction was performed onNRF2mRNA, and western blotting was performed to detect Nrf2 proteins in the nucleus or cytoplasm of RPE cells.Results. BL exposure increased cell death rate and ROS generation in ARPE-19 cells in a time-dependent manner; cell death was caused by apoptosis. Moreover, BL exposure inducedNRF2mRNA upregulation and Nrf2 nuclear translocation in RPE. Cell death rate was significantly higher in RPE cells fromNrf2−/−mice than from wild-type mice.Conclusions. The Nrf2 pathway plays an important role in protecting RPE cells against BL-induced oxidative stress.

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

PURPOSE To analyze the roles of vitreal macrophages and circulating leukocytes in retinal vascular growth. METHODS Bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P)1 mice after irradiation. The mice were exposed to 76% to 78% oxygen (P7-P12), to initiate oxygen-induced retinopathy (OIR). The eyes were collected at P8, P17, and P30, to analyze the engraftment of GFP-positive cells in the retina. GFP-positive peritoneal macrophages, clodronate liposomes, or control liposomes were injected into the eyes at P5 or P12 to examine the effects at P8 or P17. The number of Iba1-positive vitreal macrophages was quantified from histologic sections at P12 and P17. RESULTS Few transplanted GFP-positive cells were found in the retina at P8 in both wild-type and OIR mice. However, their number increased at P17 during retinal neovascularization in OIR. Most GFP-positive cells were Iba1-positive microglia, which comprised a minority of the total retinal microglia. Intravitreal injection of peritoneal macrophages showed only incidental migration of these cells into the wild-type retinas (P8), whereas the engraftment was more robust, typically around the neovascularization, in OIR mice (P17). Furthermore, native macrophages in the vitreous cavity became fewer (37.7% reduction) during neovascularization in OIR at P17. The selective depletion of vitreal macrophages by clodronate liposomes at P12 reduced retinal neovascularization in OIR mice by 59.0% at P17. CONCLUSIONS Vitreal macrophages are attracted to the site of pathologic angiogenesis triggered by retinal ischemia, where they actively participate in vascular development.

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).

Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A1 adenosine receptor (A1R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A1R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A1R, as evident from coimmunoprecipitation and BRET2 analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A1R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.

Recently, a variety of methods for protein structure prediction have been developed. However, there are only a limited number of studies where the results have been adequately validated. With this in mind, we have evaluated our previously predicted model of lactate oxidase by comparison with the recently determined crystal structure. In addition, we also analyzed our thermotolerant mutants that were designed on the basis of a predicted model. The validity of these procedures was assessed by comparing the results of rational design against the interpretation of the thermostabilization mechanism. Specifically, we analyzed lactate oxidase (LOX), a useful lactic acid sensor, as an example for validation. LOX was chosen because it has an (β/α)8 barrel (TIM barrel) fold, which constitutes the basic structural framework of numerous important enzymes. We also propose an effective modeling method and thermostabilization technique for the TIM barrel fold.

Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of alpha-1,4 glucan. From our previous study [Fujii et al., Appl. Environ. Microbiol., 71, 5823-5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 A away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.

To characterize the total retinal blood flow determined by laser speckle flowgraphy (LSFG) of healthy subjects. This prospective cross-sectional study was conducted at the Nagoya University Hospital. One hundred fifteen right eyes of 115 healthy subjects (mean age: 39.4 ± 16.1 years) were studied. The total blood flow in the retinal arteries and veins around the optic nerve head was measured separately using the total retinal flow index (TRFI), which represents blood flow volume. The lumen diameters of the retinal vessels determined by LSFG and by adaptive optics (AO) camera were compared. The images obtained by LSFG and AO camera were merged, and the distribution of the mean blur rates (MBRs), which represent the velocities of the erythrocytes, was evaluated on the images. The mean TRFI in veins (1812 ± 445, arbitral units) was significantly higher than that in arteries (1455 ± 348, arbitral units; P

To evaluate sex-related differences in ocular blood flow of healthy subjects using laser speckle flowgraphy (LSFG).In this prospective cross-sectional study, we examined 103 healthy volunteers (47 males, 56 females; mean age: 39.3 ± 15.6 years and 42.1 ± 18.7 years, respectively). The blood flow to the optic nerve head (ONH) and choroid was assessed with LSFG, including mean blur rate (MBR) and pulse waveform variables. We evaluated sex-related differences in these variables and compared them with those in other clinical parameters.A linear single regression showed that the ONH-MBR (r = -0.402, P0.001) and five ONH pulse waveforms were significantly correlated with sex. A multiple stepwise regression analysis revealed that sex (β = 0.389, P0.001) and age (β = -0.290, P = 0.002) were independent factors, indicating the ONH-MBR, age (β = -0.394, P0.001), and subfoveal choroidal thickness (β = 0.221, P = 0.016) were independent factors indicating the choroidal MBR. Moreover, sex was an independent factor indicating the five ONH pulse waveform parameters that were consistent with results of the linear single regression. The optic nerve head MBR in the female group was significantly higher than that in the male group (P0.001), but no differences were observed in the choroid between the groups (P0.05).Sex-related differences are present in ocular blood flow in the ONH, but not in the choroid in healthy subjects. We believe that these differences should be considered when interpreting blood flow data in ocular diseases.

Purpose: To improve the state-of-the-art teaching system by creating surgical videos with synchronised vertical 2-split screens. Methods: An ultra-compact, wide-angle point-of-view camcorder (HX-A1, Panasonic) was mounted on the surgical microscope focusing mostly on the surgeons' hand movements. In combination with the regular surgical videos obtained from the CCD camera in the surgical microscope, synchronised vertical 2-split-screen surgical videos were generated with the video-editing software. Results: Using synchronised vertical 2-split-screen videos, residents of the ophthalmology department could watch and learn how assistant surgeons controlled the eyeball, while the main surgeons performed scleral buckling surgery. In vitrectomy, the synchronised vertical 2-split-screen videos showed the surgeons' hands holding the instruments and moving roughly and boldly, in contrast to the very delicate movements of the vitrectomy instruments inside the eye. Conclusions: Synchronised vertical 2-split-screen surgical videos are beneficial for the education of young surgical trainees when learning surgical skills including the surgeons' hand movements.

Choroidal neovascularization (CNV) is the main pathogenesis of age-related macular degeneration (AMD), which leads to severe vision loss in many aged patients in most advanced country. CNV compromises vision via hemorrhage and retinal detachment on account of pathological neovascularization penetrating the retina. Plasma medicine represents the medical application of ionized gas "plasma" that is typically studied in the field of physical science. Here we examined the therapeutic ability of plasma-activated medium (PAM) to suppress CNV. The effect of PAM on vascularization was assessed on the basis of human retinal endothelial cell (HREC) tube formation. In mice, laser photocoagulation was performed to induce CNV (laser-CNV), followed by intravitreal injection of PAM. N-Acetylcysteine was used to examine the role of reactive oxygen species in PAM-induced CNV suppression. Fundus imaging, retinal histology examination, and electroretinography (ERG) were also performed to evaluate PAM-induced retinal toxicity. Interestingly, HREC tube formation and laser-CNV were both reduced by treatment with PAM. N-acetylcysteine only partly neutralized the PAM-induced reduction in laser-CNV. In addition, PAM injection had no effect on regular retinal vessels, nor did it show retinal toxicity in vivo. Our findings indicate the potential of PAM as a novel therapeutic agent for suppressing CNV.

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 degrees C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution.

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of α-1,4 glucans to produce cyclic α-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.

Amylomaltase (EC 2.4.1.25) from Thermus aquaticus catalyzes an intramolecular transglycosylation of α-1,4 glucan and produces cycloamylose, which is a cyclic α-1,4 glucan with a degree of polymerization of 22 and higher. The amylomaltase has weak but significant hydrolytic activity together with its major transglycosylation activity, which consequently decreases the yield of cycloamylose. To diminish the hydrolytic activity of this enzyme, random mutations are introduced into the gene coding for this enzyme. In the random mutagensesis experiment, it is suggested that tyrosine 54 (Y54), far away from the catalytic site, was involved in hydrolytic activity. In order to investigate the function of Y54, we have performed saturating mutagenesis at Y54 within the amylomaltase and examined the properties of the mutated enzymes. The reaction specificities of the mutated enzymes were surprisingly changed by only one amino acid replacement at Y54. Y54G mutated enzyme had higher cyclization activity in addition to the lower hydrolytic activity. These mutated enzymes also provided useful information to gain further understanding for the activity and the specificity of this enzyme.

We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (beta/alpha)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 degrees C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.

Many biologically active heterodimeric proteins of snake venom consist of two C-type lectin-like subunits. One of these proteins, habu IX/X-bp, is a Gla domain-binding protein whose subunits both bind to a Ca2+ ion, with a total of two Ca2+-binding sites. The molecular modeling and Ca2+-binding analysis of echis IX/X-bp revealed that it lacks one of two Ca2+-binding sites, though the folding of this subunit is conserved. It is concluded that heterodimeric C-type lectin-like proteins function independent of Ca2+ and have essentially a similar folding to habu IX/X-bp.

Even with a high surgical success rate for retinal detachment and proliferative vitreoretinopathy (PVR) supported by the robust improvement in vitrectomy surgery and its related devices, certain questions still remain for the pathogenesis and treatment of PVR. One of the important biological events in PVR is epithelial-mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells. MicroRNAs are noncoding, small, single-strand RNAs that posttranscriptionally regulate gene expression and have essential roles in homeostasis and pathogenesis in many diseases. Recently, microRNAs also had a critical role in EMT in many tissues and cells. One main purpose of this brief review is to describe the knowledge obtained from microRNA research, especially concerning vitreoretinal diseases. In addition, the potential role of microRNAs in prevention of PVR by regulating EMT in RPE cells is described. Understanding microRNA involvement in PVR could be helpful for developing new biological markers or therapeutic targets and reducing the rate of visual disability due to PVR.

Polypoidal choroidal vasculopathy (PCV) is characterized by polyp-like choroidal neovascularization and a branching vascular network. Intravitreal aflibercept injection (IAI) or photodynamic therapy (PDT) is used for treatment. We retrospectively compared the 1-year outcomes of IAI monotherapy and its combination with initial PDT for PCV. Twelve eyes with naïve PCV received three IAIs and a single PDT after the first IAI and as needed injection (combination group); 11 eyes with naïve PCV received three IAIs and as needed injections (IAI group). Significant improvements in visual acuity after 2 months and in CRT after 1 month were maintained at 12 months in both groups (both P < 0.05); groups did not differ significantly at any time point. CCT significantly reduced after 3 and 12 months in the combination group (both P < 0.05) but not in the IAI group. A mean of 3.7 ± 0.9 and 5.6 ± 2.0 injections was administered to the combination and IAI groups, respectively (P = 0.013). Within a 1-year period, combination therapy was found to yield similar visual acuity and retinal structure improvements and maintenance as IAI monotherapy while requiring fewer IAIs.

Significance Geographic atrophy is a late stage of age-related macular degeneration (AMD) that causes blindness in millions worldwide characterized by death of the retinal pigmented epithelium (RPE). We previously reported that RPE death is due to a deficiency in the enzyme DICER1, which leads to accumulation of toxic Alu RNA. We also demonstrated that Alu RNA causes RPE death by activating an immune platform called the NLRP3 inflammasome. However, the precise mechanisms of RPE death in this disease remained unresolved. The present study indicates that Alu RNA induces RPE death by activating the enzyme Caspase-8 downstream of inflammasome activation and that blocking Caspase-8 rescues RPE degeneration. This implicates apoptosis as the cell death pathway responsible for Alu RNA cytotoxicity, and these findings provide new potential therapeutic targets for this disease.

Purpose To examine the effectiveness of interleukin-18 (IL-18) on choroidal neovascularization (CNV) and retinal pigment epithelium (RPE) in humans and mice. Methods Serum IL-18 levels in patients with wet and dry AMD who were older than 50 years were measured and compared with those of age-matched controls. In mice, laser photocoagulation was performed in the retina to induce experimental CNV, and CNV volume was measured in eyes injected with recombinant IL-18 (rIL-18) and IL-18 neutralizing antibody (nIL-18Ab) compared with those injected with control. Tube formation assay was performed on human retinal endothelial cells (HREC) with rIL-18 administration in vitro. After subretinal injection of rIL-18, fundus change in the injected eyes was evaluated; active caspase-3 level was measured in the RPE/choroid complex, and tight junction integrity in RPE was visualized by zonula occludens-1 (ZO-1) staining. Results Serum IL-18 levels in dry AMD patients were higher than those in control. Mouse rIL-18 or nIL-18Ab did not induce significant change in CNV volume compared with controls or change tube formation in HREC. Subretinal injection of rIL-18 induced retinal degeneration in the mice fundus; ZO-1 staining showed considerably disturbed RPE structure, and active caspase-3 expression was significantly higher after rIL-18 induction. Conclusions Interleukin-18 did not show a pro- or antiangiogenic effect on mouse laser-induced CNVs (laser-CNVs), whereas it directly induced RPE cell apoptosis in the mouse eye. Our results suggested that IL-18 is associated with dry AMD, but not with wet AMD.

To evaluate the refractive characteristics of adults diagnosed with retinopathy of prematurity (ROP) treated with ablation treatment as children, we measured best corrected visual acuity (BCVA, logMAR), spherical equivalent refraction (SER), axial length (AL), lens thickness (LT), anterior chamber depth (ACD) and the corneal curvature radius (CCR) from 46 eyes, 24 patients (15-30 years old) that were diagnosed with ROP. Patients were divided into two groups dependent on the size of the treated retina at the time of ablation treatment; i.e., 360 degrees group (treatment over the whole circumference of the retina; n = 18) and partial group (treatment over part of the retina; n = 28). The study showed that LT was significantly larger (P1x10(-4)) and ACD was significantly shorter (P1 x 10(-3)) in 360 degrees group (4.26 +/- 0.40 mm and 2.92 +/- 0.48 mm, respectively) than those in partial group (3.71 +/- 0.34 mm and 3.42 +/- 0.26 mm, respectively). However, there were no differences in SER (-6.52 +/- 3.54 diopter vs. -5.95 +/- 4.12 diopter, P = 0.31), AL (23.9 +/- 1.42 mm vs. 25.0 +/- 21.48 mm, P = 0.08) and CCR (7.59 +/- 0.37 mm vs. 7.59 +/- 0.19 mm, P = 0.86). These results indicated that the eyes in the 360 degrees group had larger LTs but did not have extended ALs compared with the partial group.

The access of bone morphogenetic protein (BMP) to the BMP receptors on the cell surface is regulated by its antagonist noggin, which binds to heparan-sulfate proteoglycans on the cell surface. Noggin is encoded by NOG and mutations in the gene are associated with aberrant skeletal formation, such as in the autosomal dominant disorders proximal symphalangism (SYM1), multiple synostoses syndrome, Teunissen–Cremers syndrome, and tarsal–carpal coalition syndrome. NOG mutations affecting a specific function may produce a distinct phenotype. In this study, we investigated a Japanese pedigree with SYM1 and conductive hearing loss and found that it carried a novel heterozygous missense mutation of NOG (c.406C > T; p.R136C) affecting the heparin-binding site of noggin. As no mutations of the heparin-binding site of noggin have previously been reported, we investigated the crystal structure of wild-type noggin to investigate molecular mechanism of the p.R136C mutation. We found that the positively charged arginine at position 136 was predicted to be important for binding to the negatively charged heparan-sulfate proteoglycan (HSPG). An in silico docking analysis showed that one of the salt bridges between noggin and heparin disappeared following the replacement of the arginine with a non-charged cysteine. We propose that the decreased binding affinity of NOG with the p.R136C mutation to HSPG leads to an excess of BMP signaling and underlies the SYM1 and conductive hearing loss phenotype of carriers.

Up to 50 million people worldwide are afflicted with the devastating blinding disease age-related macular degeneration (AMD)1–3. The vast majority of patients have the currently untreatable “dry” or atrophic form of AMD, characterized by NLRP3 inflammasome-driven degeneration of the retinal pigment epithelium (RPE) supportive cell layer4,5. Blockade of the NLRP3 inflammasome is a next-generation therapeutic target in dry AMD; however, it was recently reported that inflammasome-mediated production of IL18 potentially safeguards the retina against the other, often more visually devastating form of AMD, for which dry AMD patients are at greatly increased risk of developing, known as choroidal neovascularization (CNV)6. Therefore, it is essential, prior to initiating inflammasome-targeting clinical trials, to directly and rigorously assess whether modulating IL18 or the NLRP3 inflammasome affects CNV and RPE cell health.

Protein Kinase C (PKC) is a family of regulatory enzymes. It is considered that binding with phorbol ester which are PKC activators, increases affinity of PKC for the membranes and consequently induces its conformation change. Electrostatic interactions between PKC and the membrane is assumed to be important, and performed ab initio MO calculations of one domain of PKC consisting of 50 amino acids and its complex with the ester is performed to investigate how the electrostatic potential of PKC changes through docking with the substrate. From the calculation, it is shown that the electrostatic potential of PKC near the binding site is dramatically affected through the binding, suggesting attractive interactions with the cell membrane.

To simulate lighting systems, we developed a rough surface ray-tracing (RSRT) model on the basis of the facet model. The model needs to conserve energy to analyze how much light is lost in each component in the lighting system. Though a single scattering rough surface model with a shadowing/masking function successfully describes the scattering distribution, masking output light violates the energy conservation law because it lacks multiple scattering. We thus developed an advanced RSRT (A-RSRT) model satisfying the energy conservation law that includes a shadowing/masking effect as a consequence of multiple scattering. To determine the accuracy, we set up a real shape (RS) model that outputs rigorous simulation results, although this RS model was impractical. The comparisons between the calculated results of the A-RSRT and RS models revealed that the A-RSRT model satisfied the energy conservation law and reproduced the scattering distribution precisely.

We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 α-amylase (Ba-S) and the raw starch-binding domain (domain E) of Bacillus A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy.

Small-angle X-ray scattering profiles for an amylose fragment (maltoheptaose) in aqueous solution were observed and compared with the theoretical profiles calculated for an ensemble of chain conformations generated by molecular dynamics simulations and Monte Carlo simulations. The Monte Carlo results based on the disaccharide conformation energy map obtained without explicitly considering surrounding water molecules were in satisfactory agreement with the experimental results, provided that the effective dielectric constant was set to four. In contrast, the results of the fully solvated molecular dynamics simulations performed using the Cff91, Cff, Gromos, Glycam93, and Glycam99 force-fields were unexpectedly different from each other. Among them, Cff91 gave most satisfactory agreement with experiment, but the other fields yielded conformations that were somewhat or highly extended. It was also shown that recently developed Glycam99 is a significant improvement over Glycam 93. The representative snapshots...